Membrane proteins constitute about 25-30% of the proteome of an organism and participate in basic energy metabolisms such as respiration or photosynthesis, communication between a cell and a cell or between a cell and the outside, material transfer, lipid metabolism, etc. In addition, it was reported that about 50% of commercially available drugs act on a G-protein coupled receptor (GPCR), a kind of membrane proteins, as a working point (Lundstrom, K., Bioorg. Med. Chem. Lett., 15:3654, 2005), and working points of ¼ of the top-selling 100 drugs are GPCR (Klabunde, T. and Hessler, G., ChemBioChem. 3:928-944, 2002).
However, researches for the functions and structures of membrane proteins fall behind those of water-soluble proteins although membrane proteins are economically important. This is because, unlike water-soluble proteins, it is almost impossible to produce membrane proteins, especially multipass transmembranes, by recombinant DNA techniques (Mancia F. and Hendrickson W. A, Mol. BioSyst. 3:723-734, 2007).
Therefore, unlike water-soluble proteins, it is extremely unusual to express membrane proteins by using microorganisms and, moreover, the amount of expressed membrane proteins is very small (Marullo, S. et al., Proc. Natl. Acd. Sci. USA., 85:7551, 1988; Grisshammer et al. Biochem J., 295:571, 1993). It was reported that about 3 mg per 100 g of E. coli cells were obtained through expression of fusion form of a neurotensin receptor and a maltose binding protein, which is the especially successful case (White, J. F., et al. FEBS Lett. 564:289, 2004).
However, when the expression of foreign membrane protein is induced through E. coli, hosts become dead before the expression of the target protein is observed. In order to solve this problem, the mutant E. coli C41 and C43 were developed, which do not die due to inducing expression of membrane protein after introducing a membrane protein expression vector (Miroux, B. and Walker, J. E., J. Mol. Biol., 260:289-298, 1996), and the E. coli C41 (DE3) and C43 (DE3) had been used for expression of a membrane protein (Korepanova, A., et al., Protein Science, 14:148-158, 2005).
In addition, it was reported that multi-membrane proteins of eukaryotic cells can be expressed by using proteins of Bacillus subtilus, called as Mistic, as a fusion partner and, however, it was not effective in expression of membrane proteins (Roosild T. P. et al., Science, 307:1317-1321, 2005; Wagner et al., Trends in Biotech., 24:364-371, 2006). Recently, human membrane proteins, such as occluding, claudin 4, ferric reductase and potassium channel, were expressed by using E. coli GlpF (glycerol-conducting channel protein) as a fusion partner and, however, this method cannot be applicable when an amino end of a target protein is outside a cell membrane and, in addition, the amount of expression was very small (Neophytou, I. et al., Appl. Microbiol. Biotechnol., 77:375-381, 2007).
Moreover, development of an expression system by using yeasts which have well-developed intracellular membrane systems, has been attempted. Recently, a method for deciding whether or not a membrane protein is expressed by checking the fluorescence of green fluorescent protein (GFP) after inserting a target protein, as a fusional protein with GFP, into a yeast expression vector by using GFP as an expression reporter, was developed (Osterberg M. et al., Proc. Natl. Acad. Sci., 103:11148-11153, 2006; Newstead S. et al., Proc. Natl. Acad. Sci., 104:13936-13941, 2007). In this case, the expression rate of proteins derived from animals including a human was very low and the amount of expression thereof was also very small, while the expression rate of yeast-derived proteins was high.
The present inventors has researched into a method for effective expression of membrane proteins of eukaryotic and prokaryotic cells and finally completed the present invention, a method for effective expression of a target membrane protein by combining Cystovirus phi12, a fusion partner, with a major envelope protein P9.